In vivo imaging of autophagy in a mouse stroke model

Recent studies have suggested that autophagy is involved in a neural death pathway following cerebral ischemia. In vivo detection of autophagy could be important for evaluating ischemic neural cell damage for human stroke patients. Using novel green fluorescent protein (GFP)-fused microtubule-associated protein 1 light chain 3 (LC3) transgenic (Tg) mice, in vivo imaging of autophagy was performed at 1, 3 and 6 d after 60 min transient middle cerebral artery occlusion (tMCAO). Ex vivo imaging of autophagy, testing of the autophagy inhibitor 3-methyladenine (3-MA), estern blot analysis, immunohistochemistry, terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) and fluorescent analyses were performed on brain sections following tMCAO. In vivo fluorescent signals were detected above the ischemic hemisphere through the skull bone at 1, 3 and 6 d after tMCAO, with a peak at 1 d. Similar results were obtained with ex vivo fluorescence imaging. western blot analysis revealed maximum LC3-I and LC3-II expression at 1 d after tMCAO and fluorescence immunohistochemistry demonstrated that GFP-LC3-positive cells were primarily neuronal, not astroglial or microglial, cells. The number of GFP-LC3/TUNEL double-positive cells was greater in the peri-ischemic area than in the core. These results provided evidence of in vivo autophagy detection, with a peak at 1 d, in a live animal model following cerebral ischemia. This novel technique could be valuable for monitoring autophagic processes in vivo in live stroke patients, as well as for clarifying the detailed role of autophagy in the ischemic brain, as well as in other neurological diseases.